Quantitative analysis of chemiluminescence signals using a cooled charge-coupled device camera
Quantitative analysis of chemiluminescence signals using a cooled charge-coupled device camera
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DOI：
10.1016/j.ab.2005.07.008
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发表时间：
2005-10-01
影响因子：
2.9
通讯作者：
Gorenstein, DG
中科院分区：
文献类型：
--
作者：
Kang, J;Lee, MS;Gorenstein, DG
化学发光被认为是生命科学中放射性同位素检测的替代方法。这种非放射性同位素方法的一个应用是电泳迁移率变动分析。定量应用的基本要求是化学发光信号与浓度成线性比例。然而，化学发光的产生是一个多步骤过程，因此不能假设线性。因此，在将该方法用作定量工具之前，验证线性非常重要。我们使用了一个商业化学发光发生系统，以评估生物素标记的tRNA和单链DNA的定量测量的有效性。结果表明，化学发光信号与生物素标记核酸的量之间呈双曲线关系，而不是线性关系。然而，发现用小于50 fmol的生物素标记的核酸，其对应于20 μ l中的2.5 nM，可以在5%误差内证明线性。因此，基于荧光的定量测量在这些限制内是可靠的方法。(C)2005年爱思唯尔公司All rights reserved.
Chemiluminescence has been considered an alternative to radioisotopic detection of materials in the life sciences. One application of this nonradioisotopic method is the electrophoretic mobility shift assay. The essential requirement for quantitative applications is that the chemiluminescence signal is linearly proportional to the concentration. However, the generation of chemiluminescence is a multi-step process, therefore linearity cannot be assumed. Therefore, it is important to verify linearity before applying the method as a quantitative tool. We used a commercial chemiluminescence generating system to evaluate the validity of quantitative measurements of biotin-labeled tRNA and single-stranded DNA. The results indicate that the relationship between the chemiluminescence signals and the quantity of biotin-labeled nucleic acids is hyperbolic rather than linear. However, it was found that with less than 50 fmol of biotin-labeled nucleic acid, which corresponds to 2.5 nM in 20 mu l, linearity can be demonstrated within 5% error. Therefore, chemiluminescence-based quantitative measurements are a reliable method within these limitations. (C) 2005 Elsevier Inc. All rights reserved.